Non-reduced SDS-PAGE showed no bands originated from aggregates in the native protein sample. The height of the most prominent band in line 2 of the gel (Figure 5-1, A) is around 160 kDa and thus originates from the monomer. No species larger than the monomer and its isoforms are detected in SDS-PAGE. Two light bands referring to species smaller than 100 kDa are detected in the native sample as well. They resemble fragments of the antibody that were generated by the harsh conditions of denaturing SDS-PAGE. Comparing the resulting pattern of native and light exposed sample, they look similar in the majority of bands, though the intensity of the bands differs. However, after light exposure additional stains above the 160
93
kDa band arise. These weak areas probably derived from covalently aggregates within the sample, though no clear band can be detected.
Figure 5-1 – Silver-stained SDS-PAGE gels of huAb.
A) SDS-PAGE under non-reducing conditions B) SDS-PAGE under reducing conditions
In line 1 the resulting bands of the HiMark™ Unstained Protein Standard are shown and some dedicated molecular weight values are labelled on the left side. Line 2 represents the native huAb molecule and line 3 represent the huAb after 48 h of light exposure.
Under reducing conditions (Figure 5-1, B) the native huAb sample is completely cut in pieces through disulfide-cleavage, resulting in one intense band around 50 kDa. Instead, the light exposed sample reveals three additional bands of larger molar mass, that derived from new formed species within the sample and that are not linked by disulfide bonds.
To confirm the formation of aggregates the sample were analyzed by SEC as well. The chromatograms obtained of a single run of the native or stressed sample by SEC are shown in Figure 5-2. 200 µg of each material were injected into the system. For better comparison of the distribution of the species, the chromatogram of the native huAb is displayed in another scale.
The area under the curve (AUC) of the UV 280 nm chromatogram was used to calculate the percentage of the different species separated by SEC. The native huAb (see Figure 5-2, black line) results in one sharp peak eluting after 32 minutes. The recovery determined by Astra software was 97.0 % and the molecular weight determined by a 3-angle MALLS detector was 166 kDa. The red line depicts the chromatogram of the stressed huAb sample, with a total recovery of 79.2 % compared to the unstressed sample. The separation resulted in 3 different species eluting prior to the monomer at 32 minutes. Referring to the molecular weight behind these peaks (see Table 5-2), they are defined as high molecular weight aggregates (HMW) eluting at 15 minutes, intermediate molecular weight aggregates (IMW) eluting between 17 and 25 minutes and dimers/trimers eluting at 27.5 minutes.
A) B)
94
Figure 5-2 – SEC separation of the human IgG1 before and after light exposure.
Time dependent UV detector voltage at 280 nm and molar mass detection by MALLS after SEC separation of light exposed huAb (red line) and native huAb (black line). The UV chromatograms are scaled on the y-axes on the left side. The red y-axis refers to the stressed sample and the black y-axis refers to the native sample. The right y-axis represents the molar mass scale for both samples.
The samples of the recombinant human IgG1 antibody after 48 h of light exposure showed four different peaks in UV 280 nm detection after field-flow fractionation (see Figure 5-3).
Figure 5-3 – AF4 separation of the human IgG1 before and after light exposure.
Time dependent UV detector voltage at 280 nm and molar mass detection by MALLS of light exposed huAb (red line) and native huAb (black line) after separation by Eclipse 2.0.
95
The area under the curve (AUC) of the UV 280 nm fractogram was used to calculate the percentage of the different species separated by AF4. The native huAb (see Figure 5-3, black line) results in one single peak eluting between 17 and 27 minutes, with a peak maximum at ~23.5 minutes. The determination of the molecular weight light scattering of the overall peak yielded in 131 kDa, which is reasonable for the expected molecular weight of ~ 144 kDa for this specific antibody. Due to the nearly monodispers composition of that sample only a limited amount of 500 µg protein was injected into the channel, since overloading would result in a poor peak shape and fronting or tailing effects. However, the slightly elevated baseline prior to the main peak is attributed to the high injection mass. The calculated total recovery was 96.8 %. To maximize the amount of protein aggregates fractionated and collected after elution, 100 µl of the sample after light exposure was injected into the system. The concentration of the sample was 10 mg/mL, thus a maximum recovery of 1 mg protein was procurable. The total recovery detected by UV spectroscopy at 280 nm was 88.4 % of protein, compared to the unstressed sample. The fractogram was subdivided into four different peaks and one shoulder shown in Figure 5-3. Using the 18-angle MALLS detector the molecular weights (MW) of these species were determined. The three major eluting peaks represent fragments of the IgG1 (elution maximum at 15.5 minutes), the monomeric protein (elution maximum at 22 minutes), and oligomeric structures (elution maximum at 33 minutes). Furthermore, a shoulder eluted directly after the monomeric species between 24.5 and 27.5 minutes. Additionally a void peak arose when analyzing this sample. The elution time of this novel peak was at 7 – 7.5 minutes.
Determination of the MW of this void peak with Astra 5.3 software was not possible. This peak most likely represents large aggregates that elute prior to all other species due to the so called steric mode of flied-flow fractionation (see Chapter 2).
The results of both orthogonal methods are summarized in Table 5-2.
Table 5-2 – Comparison of SEC and AF4 analysis of huAb after light exposure.
Overview of molecular weight determination as well as the content of the separated protein species in SEC (MWSEC, Content SEC) and in AF4 (MWAF4, Content AF4) measurements.
Protein species MW SEC MW AF4 Content SEC Content AF4
Fragments n/a 37 kDa 8.6 % 6.7 %
Monomer 162 kDa 166 kDa 35.2 % 25.4 %
Dimer / Trimer 415 kDa 295 kDa 15.6 % 9.5 %
Oligomers 1867 kDa (IMW)
6620 kDa (HMW) 2771 kDa 30.0 % (IMW)
10.6 % (HMW) 54.7 %
Total recovery --- --- 79.2 % 88.4 %
The overall recovery is pretty much comparable within both methods. After separation with AF4, 88.4 % of the original protein content was detected, whereas the analysis by SEC resulted in only 79.2 %. The difference is at least in part due to the missing void peak in SEC, representing large aggregates. However, the AUC of this peak in AF4 amounted only 3.7 %, not
96
completing the entire difference of 9 %. Since the total recovery in both methods is not 100 %, the formation of large insoluble species that precipitated prior to injection is expected. The percentages of the various protein species differ as well. Using SEC the content of fragments, monomer and small aggregates like dimers/trimers is higher than in AF4, in exchange the recovery of oligomers is lower. Comparing the determination of the molecular weights of the species very similar results were determined for the monomer peak. For the fragment peak in AF4 37 kDa were found, whereas no value was available in SEC. The comparison of molecular weight of species larger than the monomer is flawed, because of the totally differing distribution.
For example small aggregates like dimers and trimers are separated well in SEC, forming a peak. Instead, the separation by AF4 shows only a shoulder representing these species, compromising a distinct determination of MW and AUC. Anyway, the resolution in both methods is poor and does not reach the baseline within the peaks. Therefore, all values are only approximations.